Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound found in plants. Previous studies on its biological activities suggested that caffeic acid possesses anti-oxidant (Mori et al., J Clin Biochem Nutr 2009, 45:49-55; Gulcin, Toxicology 2006, 217:213-220), anti-virus (Ikeda et al., Int J Mol Med 2011, 28:595-598), anti-cancer (Rajendra-Prasad et al., Mol Cell Biochem 2011, 349:11-19) and anti-inflammatory properties (Chao et al., Nutr Metab (Lond) 2009, 6:33). Moreover, its derivative, caffeic acid phenethyl ester (CAPE), has drawn great attention because of its demonstrated therapeutic effects including its potential as an anti-diabetic and liver-protective agent as well as an anti-tumor drug for human breast cancer treatment (Wu et al., Cancer Lett 2011, 308:43-53; Celik et al., Pharmacol Res 2009, 60:270-276).
Caffeic acid is one of the pivotal intermediates of plant phenylpropanoid pathway starting from the deamination of phenylalanine which generates cinnamic acid. Followed by a two-step sequential hydroxylation at the 4- and 3-position of the benzyl ring, cinnamic acid is converted into caffeic acid via p-coumaric acid (FIG. 1; Bourgaud et al., Phytochem Rev 2006, 5:293-308; Kojima and Takeuchi, J Biochem 1989, 105:265-270). The involved enzymes, cinnamate 4-hydroxylase (C4H) and p-coumarate 3-hydroxylase (C3H) are plant-specific cytochrome P450 dependent monooxygenases. Due to their instability and membrane-bound property, the purification and characterization of these enzymes are quite challenging, particularly for C3H (Kim et al., Protein Expr Purif 2011, 79:149-155). It was also suggested that the hydroxylation at the 3-position could also occur after p-coumaric acid is esterified, which does not generate caffeic acid as the intermediate (Bourgaud et al., Phytochem Rev 2006, 5:293-308; Kneusel et al., Arch Biochem Biophys 1989, 269:455-462). Recently, genes and enzymes involved in caffeic acid biosynthesis were also reported in the actinomycete Saccharothrix espanaensis. A tyrosine ammonia lyase (TAL) encoded by sam8 and a microbial C3H encoded by sam5 are responsible for the conversion of tyrosine to p-coumaric acid and then to caffeic acid, respectively (Berner et al., J Bacteriol 2006, 188:2666-2673).
Currently, caffeic acid is produced by extraction from plant sources, such as coffee beans. Chemical or enzymatic hydrolysis of caffeoylquinic acid derivatives is also employed to produce caffeic acid (Wang et al., Af J Biotechnol 2009, 8:1416-1424; Yoshimoto et al., Biosci Biotechnol Biochem 2005, 69:1777-1781). Like many other secondary metabolites, caffeic acid derivatives are usually accumulated at low levels in plants and hence the isolation of these compounds is to some extent difficult and expensive.
Over the past few decades, advances in metabolic engineering and synthetic biology have enabled the production of various plant-specific secondary metabolites in recombinant microorganisms (Yan et al., Appl Environ Microbiol 2005, 71:3617-3623; Yan et al., Appl Environ Microbiol 2005, 71:5610-5613; Yan et al., Biotechnol J 2007, 2:1250-1262; Yan et al., Biotechnol Bioeng 2008, 100:126-140). Microbial systems have been explored by some researchers as an alternative to extraction for caffeic acid production. Sachan et al. reported the co-production of caffeic acid and p-hydroxybenzoic acid in Streptomyces caeruleus using p-coumaric acid as the carbon source (Appl Microbiol Biotechnol 2006, 71:720-727). More recently, the conversion of tyrosine to caffeic acid (the titer was not reported) and ferulic acid (7.1 mg/L) in E. coli was achieved by the co-expression of the enzymes encoded by the sam5 and sam8 from S. espanaensis and an O-methyltransferase from Arabidopsis thaliana (Choi et al., J Ind Microbiol Biotechnol 2011, 38:1657-1665). However, the above-mentioned studies relied on feeding the direct precursors such as tyrosine and p-coumaric acid, which would increase the production cost and cannot be preferred for large-scale production.